Modulation of microbial synthesis of 4-etylphenol and 4-ethylphenol sulfate in behavior and disease

ABSTRACT

Some embodiments relate to genetically engineered bacterial strains for modulation of levels of the bacterial metabolite 4-ethylphenol (4EP) and its sulfated form, 4-ethylphenyl sulfate (4EPS). In some embodiments, the bacteria reduce or inhibit production of 4EP or 4EPS in the gut of a subject. The bacteria can ameliorate, delay the onset or reduce the likelihood of one or more symptoms associated with anxiety and/or autism spectrum disorder (ASD) in the subject.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57. Thepresent application claims the benefit of U.S. Provisional App. No.62/458,716, filed Feb. 14, 2017, which is hereby incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under Grant No(s).MH100556 and GM099535 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention

REFERENCE TO SEQUENCE LISTING

This application is filed with an electronic sequence listing entitledCALTE129WOSEQLIST.txt, created on Feb. 13, 2018 which is 20,975 bytes insize. The information in the electronic sequence listing is herebyincorporated by reference in its entirety.

BACKGROUND

A number of behavior disorders are known. Anxiety disorders are a classof disorders that can involve various degrees of worry, fear, anddistress that can interfere with normal function. Examples of disordersassociated with anxiety include depression, chronic illness, eatingdisorders, headaches, hoarding disorder, irritable bowel syndrome, sleepdisorders, substance use disorders, attention deficit/hyperactivedisorders, chronic pain, and fibromyalgia. Autism spectrum disorders(ASD) are a group of neurodevelopmental conditions with a broad range ofmanifestations involving altered social communication and interaction,as well as repetitive, stereotyped behaviors. The prevalence of ASD inthe US, as of 2012, is 14.6 per 1,000 children (Christensen et al.,2016), with 1 million cases currently and evidence that diagnoses arerising (Fombonne, 2009; King and Bearman, 2009). ASD heritability hasbeen estimated as high as 83%, and the non-shared environmentalinfluence at 17% (Sandin et al., 2017).

Metabolites produced by gut bacteria have significant effects on hosthealth. In various disease states, the levels and localization of thesebacterial-derived molecules can fluctuate. Described herein areexperiments demonstrating direct effects of the bacterial metabolite4-ethylphenol (4EP) and its sulfated form, 4-ethylphenyl sulfate (4EPS)on symptoms of behavioral disorders such as ASD and anxiety, andembodiments related to compositions, methods, and uses for improvingbehavioral performance, for example in anxiety and/or ASD.

FIELD

Some embodiments herein relate to genetically engineered bacterialstrains for modulation of levels of the bacterial metabolite4-ethylphenol (4EP) and its sulfated form, 4-ethylphenyl sulfate (4EPS).In some embodiments, the bacteria reduce or inhibit production of 4EP or4EPS in the gut of a subject, and ameliorate, delay the onset of orreduce the likelihood of one or more symptoms associated with anxietyand/or ASD in the subject.

SUMMARY

Some embodiments include a composition or product combination comprisinga genetically engineered Bacteroides ovatus comprising aloss-of-function mutation in a gene encoding BO1194, and/or agenetically engineered Lactobacillus plantarum comprising a loss-offunction mutation in a gene encoding phenolic acid decarboxylase (PAD).In some embodiments, the composition or product combination according toany of the foregoing or following is formulated for oral or rectaladministration to a mammalian subject. In some embodiments, thecomposition or product combination according to any of the foregoing orfollowing is selected from the group consisting of: a probioticcomposition or product combination, a pharmaceutical composition orproduct combination, a dietary supplement, and a food; or a combinationof two or more of the listed items. In some embodiments, the compositionor product combination according to any of the foregoing or followingfurther comprises a polyphenol. In some embodiments, the polyphenolcomprises quercetin and/or curcumin. In some embodiments, thecomposition or product combination according to any of the foregoing orfollowing comprises the genetically engineered Bacteroides ovatus. Insome embodiments, for the composition or product combination accordingto any of the foregoing or following, the loss-of-function mutation inthe gene encoding BO1194 comprises a deletion, insertion, substitution,rearrangement, or frameshift. In some embodiments, the composition orproduct combination according to any of the foregoing or followingcomprises the genetically engineered Lactobacillus plantarum. In someembodiments, for the composition or product combination according to anyof the foregoing or following, the loss-of-function mutation in the geneencoding PAD comprises a deletion, insertion, substitution,rearrangement, or frameshift. In some embodiments, the composition orproduct combination according to any of the foregoing or followingcomprises the genetically engineered Bacteroides ovatus and thegenetically engineered Lactobacillus plantarum together in a singlecomposition. In some embodiments, the composition or product combinationaccording to any of the foregoing or following comprises the geneticallyengineered Bacteroides ovatus and the genetically engineeredLactobacillus plantarum is separate compositions. In some embodiments,for the composition or product combination according to any of theforegoing or following, the genetically engineered Bacteroides ovatus isdeficient in the production of p-coumaric acid. In some embodiments, forthe composition or product combination according to any of the foregoingor following, the genetically engineered Lactobacillus plantarum isdeficient in the production of 4-vinylphenol. In some embodiments, thecomposition or product combination according to any of the foregoing orfollowing comprises the genetically engineered Bacteroides ovatuscomprising the loss-of-function mutation in the gene encoding BO1194,and comprises no more than 10⁶ cfu of Lactobacillus plantarum. In someembodiments, the composition or product combination according to any ofthe foregoing or following comprises the genetically engineeredLactobacillus plantarum comprising the loss-of-function mutation in thegene encoding PAD, and comprises no more than 10⁶ cfu of Bacteroidesovatus, for example no more than 10⁶, 10⁵, 10⁴, or 10³ cfu. In someembodiments, the composition or product combination according to any ofthe foregoing or following comprises at least 10⁴ cfu of the geneticallyengineered Bacteroides ovatus, for example, at least 10⁴ cfu, 10⁵ cfu,or 10⁶ cfu. In some embodiments, the composition or product combinationaccording to any of the foregoing or following comprises at least 10⁴cfu of the genetically engineered Lactobacillus plantarum, for example,at least 10⁴ cfu, 10⁵ cfu, or 10⁶ cfu. In some embodiments, thecomposition or product combination according to any of the foregoing orfollowing comprises at least 10⁴ cfu of the genetically engineeredBacteroides ovatus and at least 10⁴ cfu of the genetically engineeredLactobacillus plantarum, for example, at least 10⁴ cfu, 10⁵ cfu, and/or10⁶ cfu of each. In some embodiments, the composition or productcombination according to any of the foregoing or following furthercomprises Bacteroides fragilis and/or Bacteroides thetaiotaomicro. Insome embodiments, the composition or product combination according toany of the foregoing or following is for use in ameliorating, delayingthe onset of, or decreasing the likelihood of a symptom associated withanxiety and/or autism spectrum disorder (ASD) in a subject. In someembodiments, for the composition or product combination for useaccording to any of the foregoing or following, the use comprisesinhibiting production of 4-ethylphenol (4EP) and/or 4-ethylphenylsulfate (4EPS) in the subject, thus ameliorating, delaying the onset of,or decreasing the likelihood of the symptom associated with anxietyand/or ASD. In some embodiments, for the composition or productcombination according to any of the foregoing or following, the usecomprises ameliorating, delaying the onset of, or decreasing thelikelihood of the symptom associated with anxiety. In some embodiments,for the composition or product combination according to any of theforegoing or following, the use comprises ameliorating, delaying theonset of, or decreasing the likelihood of the symptom associated withASD. In some embodiments, for the composition or product combinationaccording to any of the foregoing or following, the subject is selectedas being within a class of subjects that should receive the composition.In some embodiments, for the composition or product combinationaccording to any of the foregoing or following, the subject isdetermined to have anxiety, or a level of 4EPS in a sample of thesubject is higher than that of a non-anxiety or non-ASD control subject,thus selecting the subject as being within the class of subjects thatshould receive the composition.

Some embodiments include a composition comprising quercetin and/orcurcumin for use in ameliorating, delaying the onset of, or decreasingthe likelihood of a symptom associated with anxiety and/or ASD in asubject. In some embodiments, the use according to any of the foregoingor following comprises inhibiting production of 4-ethylphenyl sulfate(4EPS) in the subject, thus ameliorating delaying the onset of, ordecreasing the likelihood of a symptom associated with anxiety. In someembodiments, for the composition for use according to any of theforegoing or following (and comprising quercetin and/or curcumin), thesubject is selected as being within a class of subjects that shouldreceive the composition. In some embodiments, for the composition foruse according to any of the foregoing or following (and comprisingquercetin and/or curcumin), (i) the subject is determined to have asymptom associated with anxiety, or (ii) a level of 4EPS in a sample ofthe subject is higher than that of a non-anxiety control subject, thusselecting the subject as being within a class of subjects that shouldreceive the composition.

Some embodiments include a method of reducing or inhibiting productionof 4-ethylphenol (4EP) and/or 4-ethylphenyl sulfate (4EPS) in a subjectin need thereof. The method can comprise administering to the subject acomposition or product combination comprising a genetically engineeredBacteroides ovatus comprising a loss-of-function mutation in a geneencoding BO1194, and/or a genetically engineered Lactobacillus plantarumcomprising a loss-of-function mutation in a gene encoding phenolic aciddecarboxylase (PAD) (for example, a product or composition as describedherein). The method can include permitting the genetically engineeredBacteroides ovatus and/or the genetically engineered Lactobacillusplantarum to proliferate in a gastrointestinal tract of the subject.Thus, production of 4EP and/or 4EPS in the subject is reduced orinhibited. In some embodiments, for the method according to any of theforegoing or following, reducing the levels of 4EP and/or 4EPSameliorates, delays the onset of, or decreases the likelihood of asymptom associated with anxiety and/or ASD in the subject. In someembodiments, the method according to any of the foregoing or followingfurther comprises, prior to said administering, selecting the subject asbeing within a class of subjects that are in need of receiving thecomposition or product combination. In some embodiments, for the methodaccording to any of the foregoing or following, the selecting comprisesidentifying the subject as having a symptom associated with anxietyand/or ASD. In some embodiments, for the method according to any of theforegoing or following, the selecting comprises detecting a level of 4EPand/or a level of 4EPS in a sample derived from the subject. In someembodiments, the method according to any of the foregoing or followingfurther comprises administering a polyphenol to the subject. By way ofexample, the polyphenol can comprise quercetin and/or curcumin. In someembodiments, for the method according to any of the foregoing orfollowing, the composition or product combination comprises thegenetically engineered Bacteroides ovatus. In some embodiments, for themethod according to any of the foregoing or following, theloss-of-function mutation in the gene encoding BO1194 comprises adeletion, insertion, substitution, rearrangement, or frameshift. In someembodiments, for the method according to any of the foregoing orfollowing, the composition or product combination comprises at least 10⁴cfu of the genetically engineered Bacteroides ovatus for example, atleast 10⁴ cfu, 10⁵ cfu, or 10⁶ cfu. In some embodiments, for the methodaccording to any of the foregoing or following, the composition orproduct combination comprises no more than 10⁶ cfu of Lactobacillusplantarum, for example no more than 10⁶, 10⁵, 10⁴, or 10³ cfu. In someembodiments, for the method according to any of the foregoing orfollowing, the composition or product combination comprises thegenetically engineered Lactobacillus plantarum. In some embodiments, forthe method according to any of the foregoing or following, theloss-of-function mutation in the gene encoding PAD comprises a deletion,insertion, substitution, rearrangement, or frameshift. In someembodiments, for the method according to any of the foregoing orfollowing, the composition or product combination comprises at least 10⁴cfu of the genetically engineered Lactobacillus plantarum for example,at least 10⁴ cfu, 10⁵ cfu, or 10⁶ cfu. In some embodiments, for themethod according to any of the foregoing or following, the compositionor product combination comprises no more than 10⁶ cfu of Bacteroidesovatus, for example no more than 10⁶, 10⁵, 10⁴, or 10³ cfu. In someembodiments, for the method according to any of the foregoing orfollowing, the composition or product combination comprises thegenetically engineered Bacteroides ovatus and the genetically engineeredLactobacillus plantarum. In some embodiments, for the method accordingto any of the foregoing or following, the genetically engineeredBacteroides ovatus and the genetically engineered Lactobacillusplantarum are administered in a single composition. In some embodiments,for the method according to any of the foregoing or following, thegenetically engineered Bacteroides ovatus and the genetically engineeredLactobacillus plantarum are administered in separate compositions. Insome embodiments, for the method according to any of the foregoing orfollowing, the genetically engineered Bacteroides ovatus and thegenetically engineered Lactobacillus plantarum are administered atseparate times. In some embodiments, for the method according to any ofthe foregoing or following, the genetically engineered Bacteroidesovatus and the genetically engineered Lactobacillus plantarum areadministered at the same time. In some embodiments, for the methodaccording to any of the foregoing or following, the geneticallyengineered Bacteroides ovatus and the genetically engineeredLactobacillus plantarum are administered at separate times, or at thesame time. In some embodiments, for the method according to any of theforegoing or following, the subject is germ free. In some embodiments,for the method according to any of the foregoing or following, themethod further comprises administering an antibiotic to the subjectprior to administering the composition or product combination. Theantibiotic can reduce a total quantity of gut bacteria of the subject byat least 80% prior to the administering of the composition, for examplereducing the quantity of gut bacteria of the subject by at least 80%,85%, 90%, 95%, 97%, 98%, 99%, or 99.9%.

Some embodiments include a method of reducing or inhibiting productionof 4-ethylphenol (4EP) and/or 4-ethylphenyl sulfate (4EPS) in a subjectin need thereof, the method comprising administering an amount ofpolyphenol to the subject. In some embodiments, the polyphenol comprisesquercetin and/or curcumin. In some embodiments, the amount is effectiveto reduce and/or inhibit sulfation of the 4EP and/or 4EPS. In someembodiments, reducing the levels of 4EP and/or 4EPS ameliorates, delaysthe onset of, or decreases the likelihood of a symptom associated withanxiety in the subject. In some embodiments, the method furthercomprises, prior to the administering, selecting the subject as beingwithin a class of subjects that are in need of receiving thecomposition. In some embodiments, the selecting comprises identifyingthe subject as having a symptom associated with anxiety. In someembodiments, the selecting comprises detecting a level of 4EP and/or alevel of 4EPS in a sample derived from the subject. In some embodiments,for any method of reducing or inhibiting production of 4EP and/or 4EPSas described herein, the method, further comprising administering to thesubject a composition or product combination comprising a geneticallyengineered Bacteroides ovatus comprising a loss-of-function mutation ina gene encoding BO1194, and/or a genetically engineered Lactobacillusplantarum comprising a loss-of-function mutation in a gene encodingphenolic acid decarboxylase (PAD) (for example, a composition or productcomposition as described herein).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E are a series of graphs showing quantification and effects of4EPS on mice in accordance with some embodiments herein. FIG. 1A showsrelative quantification of 4EPS detected by GC/LC-MS. Wildtype mice wereinjected i.p. with saline or 30 mg/kg 4EPS potassium salt daily from 3to 6 weeks of age. FIG. 1B shows center durations. FIG. 1C showsdistance from center. FIG. 1D shows startle (Vmax). FIG. 1E showsprepulse inhibition (PPI)(%).

FIGS. 2A-D are a series of graphs showing 4EPS detection in human ASD inaccordance with some embodiments herein. FIGS. 2A and 2C each show a4EPS standard curve. FIG. 2B shows % detectable 4EPS in plasma samples.FIG. 2D shows % samples above lowest standard for 4EPS in plasma. “Gp”refers to general population, and “Au” refers to an ASD population.

FIGS. 3A-C are a series of schematics and graphs showing 4EP and 4EPSsynthesis of some embodiments. FIG. 3A shows the validated 4EP naturalbiosynthetic pathway. FIG. 3B shows in vitro 4EP production. FIG. 3Cshows 4EPS levels in urine for engineered bacterial pairs.

FIGS. 4A-I are a series of schematic images and graphs showingembodiments of responses to 4EPS injections. FIG. 4A show a schematic ofmovements in an elevated plus maze, and FIGS. 4B-C are graphs showingresponses in the elevated plus maze. FIG. 4D shows a schematic ofmovements in a light/dark box, and FIGS. 4E-F show responses in thelight/dark box. FIG. 4G is a schematic of movements in an open fieldarena, and FIGS. 4H-I are graphs showing responses in the open fieldarena.

FIGS. 5A-B are a series of graphs showing open field arena behavior ofmice colonized with 4EP+/− bacterial pairs of some embodiments herein.FIG. 5A shows frequency of entries in to a segment of the arena as wellas total duration of time spent there. FIG. 5B shows total distancemoved and total time spent moving.

FIGS. 6A-B are a series of graphs showing elevated plus maze (EPM)behavior of mice colonized with 4EP+/− bacterial pairs of someembodiments herein. FIG. 6A is a series of graphs showing frequency ofentries in to a segment of the maze as well as total duration of timespent there. FIG. 6B is a graph showing total distance moved.

FIGS. 7A-C are a series of graphs showing light/dark box behavior ofmice colonized with 4EP+/− bacterial pairs of some embodiments herein.Shown are frequency in light (FIG. 7A), total duration in light (FIG.7B), and latency of first entry (FIG. 7C).

FIGS. 8A-C are a series of graphs showing repetitive behavior scores ofmice colonized with engineered bacteria either lacking or producing 4EPof some embodiments herein. Shown are grooming behavior (FIG. 8A),digging behavior (FIG. 8B), and marble burying behavior (FIG. 8C).

FIGS. 9A-B are a series of graphs showing results of an ultrasonicvocalization assay of some embodiments herein. Male mice were colonizedwith engineered bacteria either lacking or producing 4EP. Shown arefrequency of ultrasonic vocalization events (FIG. 9A) and duration ofultrasonic vocalization events (FIG. 9B).

FIG. 10 is a graph showing fecal pellets produced in an open field assayfor mice colonized with engineered bacteria either lacking or producing4EP.

FIGS. 11A-E are a series of graphs illustrating analysis of sulfation of4-ethylphenol (4EP) or para-cresol (PC) in accordance with someembodiments herein. FIG. 11A is a graph illustrating the detection ofsulfation of 4EP and PC. FIG. 11B is a graph illustrating LCMS detectionof sulfated donor. FIG. 11C is a graph illustrating that Quercetininhibits SULT1A1 in vitro. FIG. 11D is a graph illustrating that SULT1A1(sulfotransferase family 1A member 1) is expressed in GI tract ofcolonized mice. FIG. 11E is a graph illustrating tissue sulfation of4EP.

FIG. 12A is a schematic diagram illustrating a B. ovatus 01194 codingsequence (SEQ ID NO: 1) of some embodiments herein. FIG. 12B is aschematic diagram illustrating a Lactobacillus plantarum phenolic aciddecarboxylase (PAD) coding sequence (SEQ ID NO: 2) of some embodimentsherein. FIG. 12C is a schematic diagram illustrating a Bacillus subtilisphenolic acid decarboxylase (PAD) coding sequence (SEQ ID NO: 5) of someembodiments herein.

DETAILED DESCRIPTION

Decreasing or inhibiting the production of 4EPS (or one or more of itsprecursors, such as 4EP, 4-vinylphenol, and/or p-coumaric acid) inaccordance with some embodiments herein can ameliorate, delay the onsetof, or lower the liklihoood of symptoms of anxiety and/or autismspectrum disorder (ASD) in a mammal in need thereof. It is reportedherein that the metabolite 4EPS is elevated in the serum of subjectsthat suffer from symptoms associated with anxiety and/or autism spectrumdisorder (ASD) (it is also noted that symptoms of ASD can sometimesinclude anxiety and its associated symptoms), including the maternalimmune activation (MIA) mouse model of anxiety disorder and autismspectrum disorder (Example 1, FIG. 1A), and in human ASD patients(Example 2, FIGS. 2A-D). Furthermore, administering 4EPS to wild-typemice induced anxiety behaviors in these mice (Examples 1, 4, FIGS. 1B-D,4D-I). 4EPS is produced by bacteria such as bacteria in the gut, and the4EPS synthesis pathway has been characterized herein. Notably, thesynthesis of 4EP (which is sulfated to become 4EPS) is not produced inany one bacteria, but rather involves two bacteria that each perform aportion of the synthesis, Bacteroides ovatus, and Lactobacillusplantarum. (Example 3, FIG. 3A). Mutant B. ovatus comprising a deletionof the gene encoding BO1194 were constructed, were deficient in 4EPSproduction, and further, inhibited production of 4EPS when used tocolonize mice (Example 3, FIGS. 3B, 3C). Moreover, overexpression ofnative BO1194 and Bacillus subtilis phenolic acid decarboxylase (PAD) inB. ovatus contributed to the production of 4EP (Example 3, FIG. 3B).Colonization of mice with the bacteria comprising the deletion of thegene encoding BO1194 improved measurements of anxiety, for example,increasing frequency in center and total duration in center, anddecreasing total duration at the wall and mean duration at the wall inan open field arena (Example 5, FIG. 5A). Furthermore, the colonizationincreased total duration in center and total duration in center, anddecreased total duration at wall and mean duration at wall in anelevated plus maze (Example 6, FIG. 6A). Furthermore, the colonizationameliorated symptoms associated with ASD (in addition to anxiety), as itsignificantly lowered total duration in light (Example 7, FIG. 7B),reduced repetitive behaviors, including grooming, digging, and marbleburying (Example 8, FIGS. 8A-C), increased ultrasonic vocalizationfrequency and duration (Example 9, FIGS. 9A-B), and lowered fecal output(Example 10, FIG. 10). Accordingly, it is contemplated that inhibiting4EP (and therefore 4EPS) synthesis in the gut bacteria, for example, byculturing a genetically engineered B. ovatus comprising aloss-of-function mutation in BO1194 and/or a genetically engineered L.plantarum comprising a loss-of-function mutation in PAD in accordancewith some embodiments herein can inhibit (e.g., reduce, or completelyblock or prevent) production of 4EPS in a mammal in need thereof. Theinhibition of 4EP (and 4EPS) synthesis can ameliorate, reduce thelikelihood, and/or delay the onset of one or more symptoms associatedwith ASD and/or anxiety in the mammal. Additionally, the polyphenolsquercetin and curcumin were shown to inhibit sulfation of 4EP, and thusinhibit the production of 4EPS (Example 11, FIGS. 11A-E). Accordingly,it is further contemplated that a composition comprising quercetinand/or curcumin is useful for inhibiting the production of 4EPS in asubject in need thereof.

It is contemplated that decreasing or inhibiting the production of 4EPS(or one of its precursors, such as 4EP, 4-vinylphenol, and/or p-coumaricacid) in accordance with some embodiments herein can ameliorate, delaythe onset of, or lower the likelihood of symptoms associated withanxiety and/or ASD in a mammal in need thereof, for example a human. Asdecreasing or inhibiting production of a precursor of 4EPS is alsounderstood to decrease or inhibit 4EPS production, it will beappreciated herein that organisms referred to “deficient in 4EPS”production (or variations of this root term) may be deficient in theproduction of a precursor of 4EPS, and/or in EPS itself. Furthermore, itis appreciated that an organism deficient in producing a precursor ofEPS (e.g., p-coumaric acid, 4-vinylphenol, and/or 4EP), will also bedeficient in producing 4EPS. Thus, in some embodiments, the productionof 4EPS can be inhibited by mutating B. ovatus and/or L. plantarum genesin the pathway that synthesizes 4EPS. In some embodiments, a compositionor product combination comprising, consisting essentially of, orconsisting of genetically engineered B. ovatus bacteria and/orgenetically engineered L. plantarum bacteria deficient in producing 4EPS(for example B. ovatus comprising a loss-of-function mutation in a geneencoding BO1194, and/or L. plantarum comprising a loss-of-function in agene encoding phenolic acid decarboxylase (PAD)) is useful for reducingor inhibiting the production of 4EPS upon administration to a subject inneed thereof. As used herein a “loss-of-function” (including variationsof this root term) encompasses both partial loss-of-function (e.g., ahypomorphic mutation), as well as complete loss-of-function (e.g., anull mutation, such as a phenotypic null mutation, or a deletion of therelevant gene). Accordingly, it is contemplated that bacteria“deficient” in producing 4EPS, as referred to herein (includingvariations of this root term), can completely fail to produce 4EPS, orcan produce 4EPS at a reduced level and/or reduced rate compared tocorresponding wild-type bacteria. Furthermore, in some embodiments, acomposition or product combination comprising, consisting essentiallyof, or consisting of a polyphenol such as quercetin and/or curcumin isuseful for inhibiting the production of 4EPS in a subject in needthereof. In some embodiments, a composition or product combinationcomprises the genetically engineered B. ovatus and/or L. plantarumbacteria deficient in producing 4EPS (as described herein) and thepolyphenol such as quercetin and/or curcumin. The composition or productcombination can be used to inhibit the production of 4EPS in a subjectin need thereof. The composition or product combination can be furtheruseful for ameliorating, delaying the onset of, and/or reducing thelikelihood of one or more symptoms associated with anxiety and/or ASD inthe subject.

Compositions and/or Product Combinations Comprising GeneticallyEngineered Bacteroides ovatus and/or Lactobacillus plantarum (and/or aPolyphenol)

It is reported herein that Bacteroides ovatus deficient in genes for 4EPsynthesis can colonize the gut of subjects with anxiety and/or ASD,produce lower 4EPS levels, and ameliorate symptoms associated withanxiety and/or ASD (See Examples 3, and 5-10, and FIGS. 3C and 5A-10).As Lactobacillus plantarum also participates in the 4EP synthesis, it isfurther contemplated that Lactobacillus plantarum deficient in genes for4EP synthesis can also colonize the gut of subjects with anxiety and/orASD, produce lower 4EPS levels, and ameliorate symptoms associated withanxiety and/or ASD. Some embodiments include a composition or productcombination comprising, consisting essentially of, or consisting of agenetically engineered Bacteroides ovatus comprising a loss-of-functionmutation in a gene encoding BO1194, and/or a genetically engineeredLactobacillus plantarum comprising a loss-of function mutation in a geneencoding phenolic acid decarboxylase (PAD). It noted that in someembodiments, the components of any of the noted compositions can beprovided separately as “product combinations” in which the componentsare provided in two or more precursor compositions, which can either becombined to form the final composition (e.g., mix bacteria with anotherbacteria and/or one or more polyphenol to arrive at a final compositioncomprising a mixture of bacteria and/or polyphenol) or used inconjunction to achieve an effect similar to the single composition(e.g., administer bacteria and one or more polyphenols to a subjectsimultaneously or sequentially). In some embodiments, the composition orproduct combination comprises an amount of the genetically engineeredBacteroides ovatus and/or Lactobacillus plantarum sufficient to colonizethe gut of, and reduce or inhibit 4EPS production in a mammaliansubject. In some embodiments, the composition or product combinationfurther comprises a polyphenol such as quercetin and/or curcumin. Insome embodiments, the composition or product combination comprises anamount of the polyphenol sufficient to reduce or inhibit 4EPS productionin the subject. In some embodiments, the composition or productcombination further comprises one or more Bacteroides bacteria (otherthan B. ovatus), such as B. fragilis, B. thetaiotaomicron, or acombination of B. fragilis and B. thetaiotaomicron. In some embodiments,any composition or product combination as described herein is for use inameliorating, delaying the onset of, or reducing the likelihood of oneor more symptoms associated with anxiety and/or ASD in a subject havinganxiety or ASD, or at risk of having anxiety or ASD. In someembodiments, any composition or product combination as described hereinis for use in ameliorating, delaying the onset of, or reducing thelikelihood of one or more symptoms associated with anxiety in a subjecthaving anxiety or at risk of having anxiety. In some embodiments, theuse is for ameliorating, delaying the onset of, or reducing thelikelihood of anxiety. In some embodiments, the use is for ameliorating,delaying the onset of, or reducing the likelihood of anxiety or one ormore disorders associated with anxiety, for example, depression, chronicillness, eating disorders, headaches, hoarding disorder, irritable bowelsyndrome, sleep disorders, substance use disorders, attentiondeficit/hyperactive disorders, chronic pain, and/or fibromyalgia. Insome embodiments, any composition or product combination as describedherein is for use in ameliorating, delaying the onset of, or reducingthe likelihood of one or more symptoms associated with ASD in a subjecthaving ASD or at risk of having ASD. Example symptoms associated withASD include, but are not limited to repetitive behavior, hyperactivity,and communication disorders. Anxiety (and its associated symptoms) canalso be associated with ASD, and anxiety itself can sometimes beconsidered symptomatic of ASD. In some embodiments, the composition orproduct combination is for use in ameliorating, delaying the onset of,or reducing the likelihood of anxiety in a subject having anxiety or atrisk of having anxiety.

In some embodiments, the subject is identified and/or selected as beingwithin a class of subjects that should receive the composition orproduct combination. In some embodiments, the subject is determined tohave one or more one or more symptoms of anxiety (e.g., elevatedstartle, or restlessness) or a disorder related to anxiety (e.g.,depression, chronic illness, eating disorders, headaches, hoardingdisorder, irritable bowel syndrome, sleep disorders, substance usedisorders, attention deficit/hyperactive disorders, chronic pain,fibromyalgia), or one or more symptoms of ASD (e.g., repetitivebehaviors, anxiety, and/or deficient communication behavior), or a levelof 4EPS in a sample of the subject is higher than that of a non-anxietyand/or non-ASD (as applicable) control subject, thus selecting thesubject as being within a class of subjects that should receive thecomposition. In some embodiments, the subject has ASD, anxiety, and/oron or more of depression, chronic illness, eating disorders, headaches,hoarding disorder, irritable bowel syndrome, sleep disorders, substanceuse disorders, attention deficit/hyperactive disorders, chronic pain,and fibromyalgia. In some embodiments, the sample of the subjectcomprises, consists essentially of, or consists of urine or serum. Insome embodiments, the composition is for use in ameliorating, delayingthe onset of, or reducing the likelihood of one or more symptomsassociated with ASD and/or anxiety in a subject having anxiety or atrisk of having anxiety. The use can comprise inhibiting production of4-ethylphenol (4EP) and/or 4-ethylphenyl sulfate (4EPS) in the subject,so as to ameliorate, delay the onset of, or decrease the likelihood ofthe symptoms associated with ASD and/or anxiety. In some embodiments,the composition is for use in ameliorating, delaying the onset of, orreducing the likelihood of one or more symptoms associated with anxietyin a subject having anxiety or at risk of having anxiety, and the usecomprises inhibiting production of 4-ethylphenol (4EP) and/or4-ethylphenyl sulfate (4EPS) in the subject, so as to ameliorate, delaythe onset of, or decrease the likelihood of the symptoms associated withanxiety. In some embodiments, the composition is for use inameliorating, delaying the onset of, or reducing the likelihood of oneor more symptoms associated with ASD in a subject having ASD or at riskof having ASD, and the use comprises inhibiting production of4-ethylphenol (4EP) and/or 4-ethylphenyl sulfate (4EPS) in the subject,so as to ameliorate, delay the onset of, or decrease the likelihood ofthe symptoms associated with ASD. In some embodiments, the subject is amammal. In some embodiments, the mammal is a human.

It is shown herein that polyphenols such as quercetin and/or curcumincan inhibit or reduce the production of 4EPS by inhibiting the sulfationof 4EP (See Example 11). Further, it is reported herein that 4EPS caninduce anxiety (See, e.g., Example 4), but that reducing or inhibit 4EPSproduction can ameliorate, delay the onset of, or reduce the likelihoodof anxiety (See Examples 5-10). As such, some embodiments include acomposition comprising a polyphenol such as quercetin and/or curcumin)for use in ameliorating, delaying the onset of, or decreasing thelikelihood of anxiety and/or ASD in a subject. In some embodiments, theuse comprises inhibiting or reducing production of 4EPS in the subject,thus ameliorating, delaying the onset of, or decreasing the likelihoodof anxiety and/or ASD. In some embodiments the composition or productcombination comprises an amount of polyphenol such as quercetin and/orcurcumin sufficient to inhibit or reduce production of 4EPS in thesubject. In some embodiments, the subject is selected as being within aclass of subjects that should receive the composition. For example, insome embodiments, the subject can be determined to have anxiety, and/orASD, and/or a level of 4EPS in a sample of the subject can be determinedto be higher than those of a non-anxiety and/or non-ASD control subject.Based on any of these (or any combination of two or more of these), thesubject can thus be selected as being within a class of subjects thatshould receive the composition. In some embodiments, the composition orproduct combination further comprises genetically engineered Bacteroidesovatus comprising a loss-of-function mutation in a gene encoding BO1194as described herein, and/or a genetically engineered Lactobacillusplantarum comprising a loss-of function mutation in a gene encodingphenolic acid decarboxylase (PAD) as described herein.

In some embodiments, the composition or product combination comprises,consists essentially of, or consists of genetically engineeredBacteroides ovatus comprising a loss-of-function mutation in a geneencoding BO1194, and/or a genetically engineered Lactobacillus plantarumcomprising a loss-of function mutation in a gene encoding phenolic aciddecarboxylase (PAD). “Genetically engineered” has its customary andordinary meaning as understood by one of skill in the art in view ofthis disclosure. It refers to an organism (such as a bacterial strain)comprising one or more induced mutations or genetic modifications suchas deletions, insertions, frameshifts, and/or rearrangements, and/orcomprising on or more genetic constructs (such as a plasmid, array, orartificial chromosome) so that the organism differs from wild-type. Insome embodiments, the composition or product combination comprises,consists essentially of, or consists of genetically engineeredBacteroides ovatus comprising a loss-of-function mutation in a geneencoding BO1194. In some embodiments, the composition or productcombination comprises, consists essentially of, or consists ofgenetically engineered Lactobacillus plantarum comprising a loss-offunction mutation in a gene encoding phenolic acid decarboxylase (PAD).In some embodiments, the composition or product combination comprises,consists essentially of, or consists of genetically engineeredBacteroides ovatus comprising a loss-of-function mutation in a geneencoding BO194, and genetically engineered Lactobacillus plantarumcomprising a loss-of function mutation in a gene encoding phenolic aciddecarboxylase (PAD). By way of example, the loss-of-function mutation ineither or both genes can comprise a deletion, insertion, substitution,rearrangement, or frameshift. In some embodiments, the geneticallyengineered Bacteroides ovatus comprising the loss-of-function mutationin the gene encoding BO1194, and the genetically engineeredLactobacillus plantarum comprising the loss-of function mutation in thegene encoding PAD can be together in a single composition. In someembodiments, the genetically engineered Bacteroides ovatus comprisingthe loss-of-function mutation in the gene encoding BO1194, and thegenetically engineered Lactobacillus plantarum comprising the loss-offunction mutation in a gene encoding PAD can be in separate compositionsthat are part of the same product combination. In some embodiments, thecomposition or product combination comprises the Bacteroides ovatuscomprising the loss-of-function mutation in the gene encoding BO1194,and the loss-of-function mutation comprises a deletion, insertion,substitution, rearrangement, or frameshift. In some embodiments, thecomposition or product combination comprises the Lactobacillus plantarumcomprising the loss-of-function mutation in the gene encoding PAD, andthe loss-of-function mutation comprises a deletion, insertion,substitution, rearrangement, or frameshift. In some embodiments, thecomposition or product combination comprises the Bacteroides ovatuscomprising the loss-of-function mutation in the gene encoding BO1194 andthe Lactobacillus plantarum comprising the loss-of-function mutation inthe gene encoding PAD, and each loss-of-function mutation comprises adeletion, insertion, substitution, rearrangement, or frameshift. Theloss of function mutations in the genes encoding BO1194 and PAD in therespective Bacteroides ovatus and Lactobacillus plantarum can be of thesame type (e.g., a deletion of each gene), or of different types (e.g.,a deletion of BO1194 and a frameshift in PAD). A non-limiting examplewild-type sequence of Bacteroides ovatus BO1194 suitable for someembodiments herein (for example, as a reference from which mutations canbe made) is provided as SEQ ID NO: 1. A non-limiting example wild-typesequence of Lactobacillus plantarum PAD suitable for some embodimentsherein (for example, as a reference from which mutations can be made) isprovided as SEQ ID NO: 2. Also, a wild-type B. subtilis PAD, an orthologof B. ovatus PAD that has been experimentally validated to function in4EP synthesis (See Example 3) is provided as SEQ ID NO: 5, and it iscontemplated that in some embodiments herein, the L. plantarum geneencoding PAD is an ortholog of B. subtilis PAD. In some embodiments, amutant BO1194 sequence comprises a deletion of at least 1 nucleotide,for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, or 150 nucleotides of SEQ ID NO: 1, including ranges between anytwo of the listed values, for example, 1-150, 2-150, 5-150, 10-150,50-150, 1-100, 2-100, 5-100, 50-100, 1-50, 2-50, 5-50, 10-50, 1-20,2-20, 5-20, 10-20, 1-10, 2-10, or 5-10. In some embodiments, a mutantBO1194 sequence comprises a point mutation in SEQ ID NO: 1, for examplean insertion of a premature stop codon in-frame. In some embodiments, amutant BO1194 sequence comprises a point mutation in SEQ ID NO: 1, forexample a frameshift mutation, for example the insertion or deletion ofa quantity of nucleotides that is not a multiple of 3 (e.g., insertionor deletion of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23,25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, or50 nucleotides). In some embodiments, a mutant PAD sequence comprises adeletion of at least 1 nucleotide, for example, 1, 2, 3 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides of SEQ ID NO:2, including ranges between any two of the listed values, for example,1-150, 2-150, 5-150, 10-150, 50-150, 1-100, 2-100, 5-100, 50-100, 1-50,2-50, 5-50, 10-50, 1-20, 2-20, 5-20, 10-20, 1-10, 2-10, or 5-10. In someembodiments, a mutant PAD sequence comprises a point mutation in SEQ IDNO: 2, for example an insertion of a premature stop codon in-frame. Insome embodiments, a mutant PAD sequence comprises a point mutation inSEQ ID NO: 2, for example a frameshift mutation, for example theinsertion or deletion of a quantity of nucleotides that is not amultiple of 3 (e.g., insertion or deletion of 1, 2, 4, 5, 7, 8, 10, 11,13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38,40, 41, 43, 44, 46, 47, 49, or 50 nucleotides). The geneticallyengineered B. ovatus comprising the mutation in BO1194, and/or thegenetically engineered L. plantarum comprising the mutation in PAD canbe made using any number of molecular biology techniques, for examplehomologous recombination with a construct comprising the mutantsequence, use of a CRISPR system, or mutagenesis in conjunction withselection or screening. It is noted that the genetically engineered B.ovatus and genetically engineered L. plantarum of product combinationsand/or compositions of some embodiments are not necessarily constructedusing the same techniques. Examples of techniques suitable for makinggenetically engineered bacteria such as genetically engineered B. ovatusand/or L. plantarum in accordance with compositions and productcombinations as described herein are provided in Green and Sambrook,Molecular Cloning: A Laboratory Manual. Cold Spring Harbor LaboratoryPress, N.Y., (4th Edition) (2012), which is hereby incorporated byreference in its entirety. In some embodiments, the geneticallyengineered B. ovatus and/or L. plantarum comprises a syntheticchromosome or portion thereof comprising the relevant mutation(s) inBO194 and/or PAD.

In some embodiments, the genetically engineered Bacteroides ovatusand/or genetically engineered Lactobacillus plantarum (and/or the pairof B. ovatus and L. plantarum) of the composition or product combinationis deficient in the production of 4EP, 4EPS, or at least on intermediateshown in FIG. 3A, for example, p-coumaric acid, and/or 4-vinylphenol. Insome embodiments, the genetically engineered Bacteroides ovatus isdeficient in the production of p-coumaric acid. In some embodiments, thegenetically engineered Bacteroides ovatus and/or genetically engineeredLactobacillus plantarum (and/or the pair of B. ovatus and L. plantarum)is deficient in the production of 4-vinylphenol.

It is shown herein that Bacteroides ovatus and Lactobacillus plantarumfunction together as a pair in the synthesis of 4EPS (See, e.g., Example3). Accordingly, in some embodiments, it is contemplated that to reduceor inhibit 4EPS production, the composition or product combinationcomprises a genetically engineered Bacteroides ovatus as describedherein, but is free or substantially free of Lactobacillus plantarum. Insome embodiments, to reduce or inhibit 4EPS production, the compositionor product combination comprises a genetically engineered Lactobacillusplantarum as described herein, but is free or substantially free ofBacteroides ovatus. As used herein, “substantially free” and variationsof this root term has its customary and ordinary meaning as understoodby one of skill in the art in view of this disclosure. It refers to acomposition and/or product combination (which may be for a use or amethod as described herein) having no more than trace amounts of asubstance (e.g., a bacteria such as Bacteroides ovatus or Lactobacillusplantarum), and/or the amount or presence of the substance having noappreciable effect (e.g., behavioral effect) on the subject. Forexample, in some embodiments, a composition, product combination, use,and/or method substantially free of a bacteria comprises no more thanabout 10⁶ colony forming units (cfu) of that bacteria, for example nomore than 10⁶ cfu, 10⁵ cfu, 10⁴ cfu, 10³ cfu, 10² cfu, or 10 cfu. Insome embodiments, the composition, product combination, use, and/ormethod comprises a genetically engineered Bacteroides ovatus comprisinga loss-of-function mutation in BO1194 as described herein, and comprisesno more than 10⁶ cfu, 10⁵ cfu, 10⁴ cfu, 10³ cfu, 10² cfu, or 10 cfu ofLactobacillus plantarum. In some embodiments, the composition, productcombination, use, and/or method comprises a genetically engineeredLactobacillus plantarum comprising a loss-of-function mutation in PAD asdescribed herein, and comprises no more than 10⁶ cfu, 10⁵ cfu, 10⁴ cfu,10³ cfu, 10² cfu, or 10 cfu of Bacteroides ovatus.

In some embodiments, the composition, product combination, use, and/ormethod comprises an amount of bacteria (e.g., genetically engineeredBacteroides ovatus and/or genetically engineered Lactobacillus plantarumas described herein) sufficient to establish a colony (e.g., a colonythat persists for at least 1, 2, 3, 4 or more weeks post-inoculation) inthe gut of a human subject when administered in a standard manner formicrobiome transplant, probiotic treatment or equivalent procedures.Such an amount of bacteria may be referred to herein as an “inoculum.”In some embodiments, the amount of bacteria in any of the compositions,product combinations, uses, or methods described herein includes atleast 10⁴ cfu, for example at least 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², or 10¹³ cfu, including ranges between any of the listedvalues, for example 10⁴-10⁸ cfu, 10⁴-10⁹ cfu, 10-10¹⁰ cfu, 10⁴-10¹¹ cfu,10⁴-10¹² cfu, 10⁴-10¹² cfu, 10⁵-10⁸ cfu, 10⁵-10⁹ cfu, 10⁵-10¹⁰ cfu,10⁵-10¹¹ cfu 10⁵-10¹² cfu, 10⁵-10¹² cfu, 10⁶-10⁸ cfu, 10⁶-10⁹ cfu,10⁶-10¹⁰ cfu, 10⁶-10¹¹ cfu, 10⁶-10¹² cfu, 10⁶-10¹² cfu, 10⁷-10⁸ cfu,10⁷-10⁹ cfu, 10⁷-10¹⁰ cfu, 10⁷-10¹¹ cfu, 10⁷-10¹² cfu, 10⁷-10¹² cfu, 10⁹cfu, 10⁸-10¹⁰ cfu, 10⁸-10¹¹ cfu, 10⁸-10¹² cfu, or 10⁸-10¹² cfu. In someembodiments, the composition, product combination, use, and/or methodcomprises a log phase (e.g., at 37° C.) of bacteria for administrationto the subject. In some embodiments, the composition, productcombination, use, and/or method comprises a stationary phase (e.g., at37° C.) of bacteria for administration to the subject. In someembodiments, the bacteria of the composition, product combination, use,and/or method are isolated bacteria. In some embodiments, thecomposition or product combination comprises at least 10⁴ cfu of thegenetically engineered Bacteroides ovatus. In some embodiments, thecomposition or product combination comprises at least 10⁴ cfu, 10⁵ cfu,10⁶ cfu, 10⁷ cfu, or 10⁸ cfu of the genetically engineered Lactobacillusplantarum. In some embodiments, the composition or product combinationcomprises at least 10⁴ cfu of the genetically engineered Bacteroidesovatus and at least 10⁴ cfu of the genetically engineered Lactobacillusplantarum (e.g., at least 10⁴ cfu, 10⁵ cfu, 10⁶ cfu, 10⁷ cfu, or 10⁸ cfuof either or both bacteria).

In some embodiments, any of the compositions and/or product combinationsdescribed herein (including those for uses and/or methods as describedherein) comprises a pharmaceutically acceptable carrier or excipient.“Pharmaceutically acceptable” carriers have their ordinary and customarymeaning as would be understood by one of skill in the art in view ofthis disclosure, and include ones which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Example “Pharmaceutically acceptable” carriers in accordance withmethods and uses and compositions and product combinations herein cancomprise, but not limited to, organic or inorganic, solid or liquidexcipients which is suitable for the selected mode of application suchas oral application and/or rectal administration, and administered inthe form of a conventional pharmaceutical preparation, such as solidsuch as tablets, granules, powders, capsules, and liquid such assolution, emulsion, suspension and the like. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution such as phosphatebuffer or citrate buffer. The physiologically acceptable carrier mayalso comprise one or more of the following: antioxidants includingascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone, amino acids,carbohydrates including glucose, mannose, or dextrins, chelating agentssuch as EDTA, sugar alcohols such as mannitol or sorbitol, salt-formingcounterions such as sodium, and nonionic surfactants such as andnonionic surfactants such as TWEEN™ surfactant, polyethylene glycol(PEG), and PLURONICS™ surfactant. Auxiliary, stabilizer, emulsifier,lubricant, binder, pH adjustor controller, isotonic agent and otherconventional additives may also be added to the carriers. In someembodiments, the composition is formulated for oral administration,rectal administration, or oral and rectal administration. In someembodiments, the composition and/or product combination comprises,consists essentially of, or consists of a probiotic.

In some embodiments, the composition or product combination isformulated for oral or rectal administration to a mammalian subject. Insome embodiments, the composition or product combination is selectedfrom the group consisting of: a probiotic composition, a pharmaceuticalcomposition, a dietary supplement, and a food, or a combination of twoor more of the listed items. The compositions and/or productcombinations of some embodiments herein are available in foods anddietary supplements (for example, but not limited to capsules, tablets,powders, and liquids). Non-limiting examples of foods containingprobiotic include dairy products such as yogurt, fermented andunfermented milk, smoothies, butter, cream, hummus, kombucha, saladdressing, miso, tempeh, nutrition bars, and some juices and soybeverages. In some embodiments, the composition or product combinationcomprises a single microorganism.

Methods of Reducing and/or Inhibiting Production of 4-Ethylphenol (4EP)and/or 4-Ethylphenyl Sulfate (4EPS) in a Subject

Some embodiments include methods of reducing and/or inhibitingproduction of 4-ethylphenol (4EP) and/or 4-ethylphenyl sulfate (4EPS) ina subject. In some embodiments, the method comprises administering anamount of a composition or product combination comprising geneticallyengineered Bacteroides ovatus and/or a genetically engineeredLactobacillus plantarum as described herein, and/or a polyphenol asdescribed herein to a subject in need of reducing and/or inhibitingproduction of 4EP and/or 4EPS.

In some embodiments, a method of reducing or inhibiting production of4-ethylphenol (4EP) and/or 4-ethylphenyl sulfate (4EPS) in a subject inneed thereof, is described. The method can comprise administering to thesubject a composition or product combination comprising a geneticallyengineered Bacteroides ovatus comprising a loss-of-function mutation ina gene encoding BO1194, and/or a genetically engineered Lactobacillusplantarum comprising a loss-of function mutation in a gene encodingphenolic acid decarboxylase (PAD). The method can comprise permittingthe genetically engineered Bacteroides ovatus and/or Lactobacillusplantarum to proliferate in a gastrointestinal tract of the subject.Thus, production of 4EP and/or 4EPS is inhibited in the subject. It iscontemplated that in accordance with embodiments described herein, anycomposition or product combination comprising (or consisting essentiallyof, or consisting of) genetically engineered Bacteroides ovatus and/orgenetically engineered Lactobacillus plantarum as described herein canbe administered to the subject in the method. In some embodiments, thegenetically engineered Bacteroides ovatus and genetically engineeredLactobacillus plantarum are administered together in a singlecomposition (e.g., a co-culture). In some embodiments, the geneticallyengineered Bacteroides ovatus and genetically engineered Lactobacillusplantarum are administered separately, for example at separate times,and/or via separate routes of administration.

The genetically engineered Bacteroides ovatus and/or geneticallyengineered Lactobacillus plantarum (or the pair of geneticallyengineered Bacteroides ovatus and genetically engineered Lactobacillusplantarum) can be deficient in one or more genes that encode products inthe 4EPS synthetic pathway (See FIG. 3A), for example comprising aloss-of-function mutation in Bacteroides ovatus BO1194 and/orLactobacillus plantarum PAD as described herein. Without being limitedby theory, it is contemplated that proliferation of genetically modifiedBacteroides ovatus and/or genetically engineered Lactobacillus plantarumdeficient in the production of 4EP (and 4EPS) can inhibit production of4EP and/or 4EPS (See, e.g., Examples 3, FIG. 3B), thus resulting inlower levels of 4EP and/or 4EPS in the host, or substantial or completeelimination of 4EP and/or 4EPS in the host. Furthermore, co-culturinggenetically modified B. ovatus strains and L. plantarum strainscollectively deficient in the 4EP (and therefore 4EPS) synthetic pathwayalleviated symptoms of anxiety (See Examples 5-7, FIGS. 5A-7C), andfurther alleviated symptoms associated with ASD, for example, reducingrepetitive behaviors, including grooming, digging, and marble burying(Example 8, FIGS. 8A-C), and improving communication behaviors, forexample increasing ultrasonic vocalization frequency and duration(Example 9, FIGS. 9A-B). As the biosynthetic pathway of 4EP (and 4EPS)in B. ovatus and L. plantarum is described herein (See, e.g., Example 3)it is further contemplated that culturing other B. ovatus, L. plantarum,and/or pairs thereof comprising mutations in the 4EP (and 4EPS)biosynthetic pathway in accordance with some embodiments herein hassimilar effects in ameliorating, decreasing the likelihood, and/ordelaying the onset of symptoms of ASD and/or anxiety. Thus, in someembodiments, the method results in amelioration, delay of onset,inhibition, or reduction in the likelihood of symptoms associated withanxiety and/or or ASD. Thus, in some embodiments, the method results inamelioration, delay of onset, inhibition, or reduction in the likelihoodof ASD, anxiety, and/or a disorder associated with anxiety as describedherein. In some embodiments, reducing the levels of 4EP and/or 4EPSameliorates, delays the onset of, or decreases the likelihood of anxietyin the subject.

Some embodiments include a method of reducing or inhibiting productionof 4-ethylphenyl sulfate (4EPS) in a subject in need thereof. The methodcan comprise administering a polyphenol to the subject. It is shownherein that polyphenols can inhibit sulfation of 4EP into 4EPS (See.Example 11). Accordingly, the amount of polyphenol can be effective ofreducing or inhibiting production of 4EPS in the subject. In someembodiments, the polyphenol comprises, consists essentially of, orconsists of quercetin and/or curcumin. In some embodiments, reducing thelevels of 4EP and/or 4EPS ameliorates, delays the onset of, or decreasesthe likelihood of a symptom associated with anxiety in the subject. Insome embodiments, reducing the levels of 4EP and/or 4EPS ameliorates,delays the onset of, or decreases the likelihood of a symptom associatedwith anxiety ASD in the subject. In some embodiments, the method furthercomprises, prior to said administering, selecting the subject as beingwithin a class of subjects that are in need of receiving thecomposition. The selecting can be as described herein. In someembodiments, the selecting comprises identifying the subject as havingone or more symptoms associated with anxiety and/or ASD as describedherein.

In some embodiments, ASD, or an ASD behavior, for example a deficientcommunication, vocalization, sensorimotor, anxiety, and/or repetitivebehavior, or a combination of two or more of these is identified usingstandard diagnostic criteria, for example in the Diagnostic andStatistical Manual of Mental Disorders, Fourth Edition (DSM-4) or FifthEdition (DSM-5). In some embodiments, the presence or absence of ASD inthe subject is determined using a behavioral test, for example at leastone of the Autism Behavior Checklist (ABC), Autism diagnosticInterview-Revised (ADI-R), childhood autism Rating Scale (CARS), and/orPre-Linguistic Autism Diagnostic Observation Schedule (PL-ADOS). Thebehavioral test can include, but is not limited to, detecting thepresence and/or extent of 1) preoccupation with one or more stereotypedand restricted patterns of interest that is abnormal in either intensityor focus, 2) inflexible adherence to specific, nonfunctional routines orrituals, c) stereotyped and repetitive motor mannerisms (such as handflapping, finger flapping etc.), and/or d) persistent preoccupation withparts of objects. Non-limiting examples of behavior that can be includedin a behavioral test and suggest a need for improving behavioralperformance in the subject under the test include: a) sensory behaviors,including poor use of visual discrimination when learning, seems not tohear, so that a hearing loss is suspected, sometimes shows no “startleresponse” to loud noise”, sometimes painful stimuli such as bruises,cuts, and injections evoke no reaction, often will not blink when brightlight is directed toward eyes, covers ears at many sounds, squints,frowns, or covers eyes when in the presence of natural light, frequentlyhas no visual reaction to a “new” person, stares into space for longperiods of time; b) relating behaviors: frequently does not attend tosocial/environmental stimuli, has no social smile, does not reach outwhen reached for, non-responsive to other people's facialexpressions/feelings, actively avoids eye contact, resists being touchedor held, is flaccid when held in arms, is stiff and hard to held, doesnot imitate other children at play, has not developed any friendships,often frightened or very anxious, “looks through” people; c) body andobject use behaviors: whirls self for long periods of time, does not usetoys appropriately, insists on keeping certain objects with him/her,rocks self for long periods of time, does a lot of lunging and darting,flaps hands, walks on toes, hurts self by banging head, biting hand,twirls, spins, and bangs objects a lot, feel, smell, and/or tasteobjects in the environment, gets involved in complicated “rituals” suchas lining things up, is very destructive; and d) language behaviors:does not follow simple commands given once, has pronoun reversal, speechis atonal, does not respond to own name when called out among twoothers, seldom says “yes” or “I”, does not follow simple commandsinvolving prepositions, gets desired objects by gesturing, repeatsphrases over and over, cannot point to more than five named objects,uses 0-5 spontaneous words per day to communicate wants and needs,repeats sounds or words over and over, echoes questions or statementsmade by others, uses at least 15 but less than 30 spontaneous phrasesdaily to communicate, learns a simple task but “forgets” quickly, strongreactions to changes in routine/environment, has “special abilities” inone area of development, which seems to rule out mental retardation,severe temper tantrums and/or frequent minor tantrums, hurts others bybiting, hitting, and/or kicking, does not wait for needs to be met,difficulties with toileting, does not dress self without frequent help,frequently unaware of surroundings, and may be oblivious to dangeroussituations, prefers to manipulate and be occupied with inanimate things,and/or a developmental delay identified at or before 30 months of age.One of ordinary skill in the art would appreciate that the attendingphysician would know how to identify a subject in need of treatmentdisclosed herein.

In some embodiments, the subject in need of reducing and/or inhibitingproduction of 4EP and/or 4EPS has one or more symptoms associated withanxiety and/or ASD. Example symptoms associated with anxiety includerestlessness and enhanced startle. Example symptoms associated with ASDinclude, but are not limited to repetitive behavior, hyperactivity,anxiety, and a communication disorder. In some embodiments, the subjectin need has anxiety. In some embodiments, the subject in need hasanxiety or a disorder related to anxiety, for example, depression,chronic illness, eating disorders, headaches, hoarding disorder,irritable bowel syndrome, sleep disorders, substance use disorders,attention deficit/hyperactive disorders, chronic pain, and fibromyalgia.In some embodiments, the subject in need has ASD.

In some embodiments, the method further comprises, prior to saidadministering, identifying and/or selecting the subject as being withina class of subjects that are in need of receiving the composition. Forexample, in some embodiments, the subject is selected as being withinthe class by identifying the subject as having anxiety. In someembodiments, the subject is selected as being within the class bydetecting a level of 4EP and/or a level of 4EPS in a sample derived fromthe subject. A level of 4EP and/or 4EPS in the sample of the subject(for example a serum sample and/or urine sample) that is higher than ina non-ASD/non-anxiety control sample can indicated that the subject isin need of treatment. Levels of 4EP and/or 4EPS can be detected by anumber of methods, for example mass spectrometry (e.g., liquidchromatography mass spectrometry, gas chromatography mass spectrometry),or immunoassays (e.g., ELISA, lateral flow assays).

It is further observed herein that polyphenols can inhibit theproduction of 4EPS (See Example 9, FIGS. 9A-B). Accordingly, in someembodiments, any of the methods or uses described herein furthercomprises administering a polyphenol to the subject. In someembodiments, the polyphenol comprises, consists essentially of, orconsists of quercetin and/or curcumin. In some embodiments, thepolyphenol is part of the composition or product combination thatcomprises the genetically modified B. ovatus and/or L. plantarum asdescribed herein. In some embodiments, the polyphenol is part of asingle composition that comprises the genetically modified B. ovatusand/or L. plantarum as described herein and the polyphenol. In someembodiments, the polyphenol is administered together with thegenetically modified B. ovatus and/or L. plantarum. In some embodiments,the polyphenol is administered separately from the genetically modifiedB. ovatus and/or L. plantarum, for example at different times, and/orvia different routes of administration.

In some embodiments, the genetically engineered Bacteroides ovatuscomprises a loss-of-function mutation in the gene encoding BO1194, andthe loss-of-function mutation comprises a deletion, insertion,substitution, rearrangement, or frameshift. In some embodiments, thegenetically engineered Lactobacillus plantarum comprises theloss-of-function mutation in the gene encoding PAD, and theloss-of-function mutation comprises a deletion, insertion, substitution,rearrangement, or frameshift.

It is noted that Bacteroides ovatus and Lactobacillus plantarumcontributes as a pair to the synthesis of 4EP(S) (See Example 3).Accordingly, in order to reduce or inhibit synthesis of 4EP(S), it canbe advantageous to administer only mutant Bacteroides ovatus asdescribed herein in the absence of Lactobacillus plantarum, and/or toadminister only mutant Lactobacillus plantarum as described herein inthe absence of Bacteroides ovatus. In some embodiments of the method oruse as described herein, the composition or product combinationadministered to the subject comprises the genetically engineeredBacteroides ovatus, and is substantially free of Lactobacillusplantarum. In some embodiments, the composition or product combinationof the method or use comprises a genetically engineered Bacteroidesovatus comprising a loss-of-function mutation in BO1194 as describedherein, and comprises no more than 10⁶ cfu, 10⁵ cfu, 10⁴ cfu, 10³ cfu,10² cfu, or 10 cfu of Lactobacillus plantarum. In some embodiments ofthe method or use as described herein, the composition or productcombination administered to the subject comprises the geneticallyengineered Lactobacillus plantarum, and is substantially free ofBacteroides ovatus. In some embodiments, the composition or productcombination of the method or use comprises a genetically engineeredLactobacillus plantarum comprising a loss-of-function mutation in PAD asdescribed herein, and comprises no more than 10⁶ cfu, 10⁵ cfu, 10⁴ cfu,10³ cfu, 10² cfu, or 10 cfu of Bacteroides ovatus.

In some embodiments of the method or use as described herein, thesubject is germ free. “Germ free” has its customary and ordinary meaningas understood by one of skill in the art in view of this disclosure. Itcan refer to a subject whose gut is substantially free of bacteria. Forexample, the subject can be germ free to facilitate recolonization ofthe subject's gut with a composition or product combination comprisinggenetically engineered B. ovatus and/or genetically engineeredLactobacillus plantarum in accordance with some embodiments herein.

In some embodiments, the method further comprises administering anantibiotic to the subject prior to administering the composition. Theantibiotic can reduce a total quantity of gut bacteria of the subject byat least 80%, for example at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 99.9%, including ranges between any of the listed values (e.g.,80%-99.9%, 80-95%, 85%-99.9%, and 85%-95%), prior to the administeringof the composition. Example antibiotics that can be administeredinclude, but are not limited to, the group consisting of: Amoxicillin,Amoxicillin/clavulanic acid (amoxicillin+clavulanic acid), Ampicillin,Benzathine benzylpenicillin, Benzylpenicillin, Cefalexin, Cefazolin,Cefixime, Cefotaxime, Ceftriaxone, Cloxacillin, Penicillin,Phenoxymethylpenicillin (penicillin V), Piperacillin/tazobactam,Procaine benzylpenicillin, Ceftazidime, Meropenem, Aztreonam,Imipenem/cilastatin, Amikacin, Azithromycin, Chloramphenicol,Ciprofloxacin, Clarithromycin, Clindamycin, Doxycycline, Erythromycin,Gentamicin, Metronidazole, Nitrofurantoin, Spectinomycin,Trimethoprim/sulfamethoxazole, Trimethoprim, Vancomycin, Clofazimine,Dapsone, Rifampicin, Ethambutol/isoniazid,Ethambutol/isoniazid/pyrazinamide/rifampicin,Ethambutol/isoniazid/rifampicin, Isoniazid,Isoniazid/pyrazinamide/rifampicin, Isoniazid/rifampicin, Pyrazinamide,Rifabutin, Rifampicin, Rifapentine, Amikacin, Bedaquiline, Capreomycin,Clofazimine, Cycloserine, Delamanid, Ethionamide, Kanamycin,Levofloxacin, Linezolid, Moxifloxacin, p-aminosalicylic acid, rifabutin,rifapentine, rifalazil, rifaximin and Streptomycin, or a combination oftwo or more of these antibiotics.

Additional Embodiments

Described herein are genes responsible for biosynthesis of 4EP, as wellas bacterial species capable of producing it. No single organism hasbeen identified as having entire 4EP synthetic pathway, but rather pairsthat together produce 4EP in vitro and in vivo. In order to sustain highlevels of 4EP for in vivo experiments, the naturally producing pair,Bacteroides ovatus and Lactobacillus plantarum has been engineered tooverproduce the pathway (See Example 3). When germ-free mice arecolonized with (1) B. ovatus overexpressing an important gene in the 4EPpathway, BO1194, along with (2) wild-type L. plantarum, the mice displayan anxiety phenotype in the same behavior tests compared to thebacterial pair with a B. ovatus BO1194 mutant.

Some embodiments include drug development to target enzymes that produce4EP.

Some embodiments include antibiotic development to specifically targetbacteria involved in 4EP production and/or control.

Some embodiments include bacterial engineering to metabolize/degrade 4EPproduced by other species.

Some embodiments include bacterial engineering to develop a strain thatcan invade health microbiota and outcompete 4EP-producing strains.

Some embodiments include drug development to inactivate 4EP/4EPSdirectly.

Some embodiments include targeting of host pathways involved inresponding to 4EP/4EPS.

Some embodiments include treatment of a subject in need thereof usingany of the techniques described herein for treating for diseases ordisorders related to anxiety, such as: depression, chronic illness,eating disorders, headaches, hoarding disorder, irritable bowelsyndrome, sleep disorders, substance use disorders, attentiondeficit/hyperactive disorders, chronic pain, and/or fibromyalgia.

EXAMPLES Example 1: Levels and Effects of 4EPS in Mice

Levels of 4EPS in wild-type mice, and the maternal immune activation(MIA) mouse model of anxiety disorder and autism spectrum disorder (ASD)were measured.

The MIA mouse model promotes leakage of intestinal byproducts across thegut epithelium in the offspring of MIA mothers. The host is incapable ofproducing 4EP, but upon crossing the intestinal barrier in to hostcirculation, 4EP is modified with a sulfate group.

Relative quantification of 4EPS detected by GC/LC-MS showed that 4EPS issignificantly altered in MIA mouse model of autism spectrum disorder(“P”) and is restored by B. fragilis treatment (“P+BF”). n=8/group (FIG.1A). In this analysis, 4EPS was 46-fold higher in MIA pups thancontrols. Accordingly, administering B. fragilis in accordance with someembodiments herein can reduce levels of 4EPS in a subject.

To measure effects of 4EPS on wild-type mice, the wild-type mice wereinjected intraperitoneally (i.p.) with saline or 30 mg/kg 4EPS potassiumsalt daily from 3 to 6 weeks of age. Anxiety-like behavior based oncenter duration (See FIG. 1B) and locomotor behavior (See FIG. 1C) weremeasured in the open field exploration assay. n=10/group. Furthermore,the potentiated startle reflex was measured in the pre-pulse inhibition(PPI) assay, and measurements were taken of startle (Vmax) (FIG. 1C) andPPI % (FIG. 1D). n=10/group. These data show that administering 4EPSsignificantly increased anxiety behaviors in wild-type mice.

Example 2: EPS Levels in and Effects in Human Children Samples

Human plasma from ASD and neurotypical children from the generalpopulation was analyzed by Liquid Chromatography Mass Spectrometry(LC/MS) for 4EPS. As shown in FIG. 2A, 4EPS levels were quantitatedusing a 4EPS standard curve. FIG. 2B shows that the percent of totalsamples in which 4EPS was detectable from ASD and general populationchildren. Additionally, samples above the lowest standard in thestandard curve are plotted, and the ASD population had higher levels of4EPS in plasma than the general population (FIG. 2C). Another analysismade was the percent of total samples in which the 4EPS level was higherthan lowest standard (FIG. 2D). The ASD population had a greaterpercentage of samples above the lowest standard (FIG. 2D). AU=Autistic,GP=general population Example 3: 4EP(S) biosynthesis and geneticengineering of bacteria

The biosynthetic pathway of 4EP in Bacteroides ovatus and Lactobacillusplantarum was determined herein, and is summarized in FIG. 3A. Thepathway involves synthetic steps in two different bacteria, Bacteroidesovatus and Lactobacillus plantarum, which together produce 4EP. InBacteroides ovatus, BO119 converts tyrosine to p-coumaric acid. InLactobacillus plantarum, PAD converts the p-coumaric acid to4-vinylphenol. Also in Lactobacillus plantarum, the 4-vinylphenol isconverted into 4EP.

It was shown that co-culturing of engineered B. ovatus and L. plantarumproduces 4EP in vitro and in vivo. Genetically modified B. ovatus wasgenerated in order to modulate 4EPS production and confirm thecontributions of individual genes to the pathway.

PAD catalyzes decarboxylation of p-coumaric acid to produce4-vinylphenol. BACOVA_01194 is involved in the biosynthesis ofp-coumaric acid. These genes were considered as candidates for beinginvolved in the synthesis of 4EP, and were tested, among othercandidates, by constructing loss-of-function mutants and overexpressionconstructs. Approximately 20 L. plantarum mutants were also constructedin an attempt to ascertain L. plantarum genes involved in the final stepof the pathway, conversion of 4-vinylphenol to 4EP (See FIG. 3A), butthe L. plantarum genes involved in this final step were not identifiedas of the time of filing.

To construct mutant B. ovatus bacteria, B. ovatus ATCC 8483ΔBAVOVA_03071 (tdk) was used for a parent strain.

For overexpression studies, chromosomal insertions were generated ofboth the native BACOVA_01194 gene (SEQ ID NO: 1), and the genesexpressing the enzyme PAD (SEQ ID NO: 5) from Bacillus subtilis. Bothgenes (BCAOVA_1194 and PAD) were inserted in tandem, using the sameplasmid, pNBU2 (SEQ ID NO: 3). This construct confers erythromycinresistance as well.

To construct the mutant B. ovatus deleted for BO1194, the followingprotocol was used. All PCR amplification was conducted using PrimeSTARMax DNA polymerase (Takara Bio) according to the manufacturer'sinstructions. Sequences of primers were shown in Table 1. Adouble-crossover recombination method previously described was used(Koropatkin et. al., 2008). Briefly, ˜1 kb DNA fragments correspondingto the upstream and downstream regions of the target gene were amplifiedfollowed by restriction digestion. These digested fragments are ligatedinto the suicide plasmid pExchange-tdk using T4 DNA ligase (New EnglandBio). Escherichia coli S17-1λ pir competent cells were transformed withthe ligated plasmid was transformed into by electroporation andtransformants were confirmed by PCR. The positive clone harboringligated plasmid was cultivated and the plasmid was prepared. It wasfurther verified by sequencing. Both B. ovatus Δtdk and E. coli 517-1λpir harboring the plasmid were cultivated and the cells were harvested.The cell pellets were washed with PBS to remove residual antibiotics andcombined them in TYG medium. The suspension was plated on BHI-blood agarmedium without any antibiotics and grown aerobically at 37° C. for 1day. The bacterial biomass was scraped up and re-suspended in TYGmedium. The suspension was then plated on BHI-blood agar mediumsupplemented with erythromycin and gentamicin and single-crossoverintegrants were selected. These strains cultured in TYG medium overnightand plated on BHI-blood agar medium supplemented with FUdR. The deletionmutant was screened by PCR amplification and further verified bysequencing. Accordingly, B. ovatus comprising a loss-of-functionmutation in accordance with some embodiments herein was constructed.

For co-culture, B. ovatus strains and L. plantarum were cultivatedanaerobically in TYG (tryptone-yeast extract-glucose) broth and MRSbroth (BD), respectively overnight at 37° C. and the cells wereharvested followed by minimal medium (MM) wash. The cell pellet wasre-suspended with MM containing 0.5 mg/ml tyrosine to be OD600 of 1.0and combined two strains in the same tube. After 1 day incubation,samples for HPLC analysis were prepared.

Multiple rounds of engineering were done to increase the yield of 4EPproduction. For example, co-cultivation of L. plantarum BAA-793 with B.ovatus +1194+PAD strain boosted 4EP level compared to B. ovatus +1194strain without the PAD inserted cocolonized with L. plantarum.

FIG. 3B illustrates 4EP detection from in vitro coculture of engineeredB. ovatus overexpressing BO1194 and PAD (chromosomal insertion ofoverexpressed BO1194, and PAD “Engineered BO” or “Eng. BO”) or theBO1194 deletion mutant (“Mutant BO”) with L. plantarum. As shown in FIG.3B, the BO1194 deletion mutant B. ovatus in accordance with someembodiments herein failed to produce 4EP in co-culture. On the otherhand the B. ovatus overexpressing BO1194 and PAD produced substantialquantities of 4EP in co-culture.

FIG. 3C illustrates quantitation of 4EPS in urine of ex-germ free micecocolonized with BO and LP strain pairs (n=5). As shown in FIG. 3C, themice co-colonized with the BO1194 deletion mutant did not have anydetectable 4EPS in their urine. On the other hand, substantialquantities of 4EPS were detected in the urine of mice co-colonized withB. ovatus overexpressing BO1194.

These experiments show that colonizing the gut of a mammalian host withB. ovatus containing a loss-of-function mutation in BO1194 impairs theproduction of 4EPS in the mammal. These experiments further show thatcolonizing the gut of a mammalian host with B. ovatus overexpressingBO1194 yields higher expression of 4EPS.

Example 4: Anxiety Behavior of Mice Injected with 4EPS

Wildtype mice were injected intraperitoneally (i.p.) with saline or 30mg/kg 4EPS potassium salt daily from 3 to 6 weeks of age and tested foranxiety in the elevated plus maze (FIG. 4A-C), light dark box (FIG.4D-F), and open field arena (FIG. 4G-I) assays.

As shown in FIGS. 4A-4C, the mice injected with 4EPS exhibited decreasedduration in the open, and decreased total terminus duration. As shown inFIGS. 4D-F, the mice injected with 4EPS exhibited decreased frequencyand total duration in the light. As shown in FIGS. 4G-I, the miceinjected with 4EPS exhibited decreased total duration in center.Distance moved was comparable to saline-injected mice.

These results indicate increasing the level of 4EPS in mice inducessymptoms associated with anxiety.

Example 5: Open Field Arena Behavior of Mice Colonized with 4EP+/−Bacterial Pairs

Germ free mice were cocolonized by gavage of 10⁸ CFU of B. ovatus eitherwith or without the 4EP biosynthetic pathway along with L. plantarum at3 weeks of age (mutants were produced as described in Example 3). Micewere tested for anxiety-like (and ASD-like) behavior in the open fieldtest at 7-8 weeks. Frequency of entries in to a segment of the arena aswell as total duration of time spent there are shown in FIG. 5A. Totaldistance moved and total time spent moving are shown in FIG. 5B.Center=innermost section of arena. Center2=section between wall andcenter.

These data show that co-colonizing mice with B. ovatus containing adeletion mutation in BO1194 (and therefore deficient in producing 4EPS)in accordance with some embodiments herein ameliorated anxiety in mice(including, for example, anxiety associated with ASD) as measured in anopen field arena, for example increasing frequency in center and totalduration in center, and decreasing total duration at wall and meanduration at wall.

Example 6: Elevated Plus Maze (EPM) Behavior of Mice Colonized with4EP+/− Bacterial Pairs

Germ free mice were co-colonized by gavage of 10⁸ CFU of B. ovatus (+/−BO1194) and L. plantarum at 3 weeks of age and tested for anxiety-likeand ASD-like behavior in the elevated plus maze test at 7-8 weeks.Frequency of entries in to a segment of the maze as well as totalduration of time spent there is shown in FIG. 6A. Total distance movedis shown in FIG. 6B. Mid-outer=outer ⅔ of open arm. Terminus=outermost ⅓of open arm. Closed=entire closed arm. Open=entire open arm.

These data show that co-colonizing mice with B. ovatus containing adeletion mutation in BO1194 (and therefore deficient in producing 4EPS)in accordance with some embodiments herein ameliorated anxiety in mice(including, for example, anxiety associated with ASD), as measured in anelevated plus maze, for example increasing total duration in center andtotal duration in center, and decreasing total duration at wall and meanduration at wall.

Example 7: Light/Dark Box Behavior of Mice Colonized with 4EP+/−Bacterial Pairs

Germ free mice were cocolonized by gavage of 10⁸ CFU of B. ovatus (+/−BO1194) and L. plantarum at 3 weeks of age and tested for anxiety-likebehavior in the light/dark box test at 7-8 weeks. Frequency of entriesin to light chamber, total duration spent in light, and the latency ofthe first entry in to light chamber are shown in FIGS. 7A-C. Frequencyin light were comparable for mice co-colonized with B. ovatus containinga deletion mutation in BO1194 (and therefore deficient in producing4EPS) and for wild-type mice (FIG. 7A), and no statistically significantdifference was observed for latency of first entry (FIG. 7C). On theother hand, the mice co-colonized with the mutant B. ovatus (andtherefore deficient in producing 4EPS) exhibited significantly lowertotal duration in light (FIG. 7B).

These data show that co-colonizing mice with B. ovatus containing adeletion mutation in BO1194 (and therefore deficient in producing 4EPS)in accordance with some embodiments herein was effective in amelioratingsymptoms associated with anxiety in mice (including, for example,anxiety associated with ASD), as measured by total duration in light ina light/dark box.

Example 8: Repetitive Behaviors of Mice Colonized with 4EP+/− BacterialPairs

Repetitive behavior scores were determined for mice colonized withengineered bacteria either lacking or producing 4EP. Mice were videorecorded during a 10 minute period of social interaction with a novelmouse. Self-grooming was scored from manual video analysis, and theresults are shown in FIG. 8A. The mice colonized with mutant bacteriadeficient in 4EPS production exhibited significantly less groomingbehavior (FIG. 8A).

Mice were placed alone in a cage with a 1 inch layer of bedding andvideo recorded for 10 minutes. Digging behavior was scored from manualvideo analysis, and the results are shown in FIG. 8B. The mice colonizedwith mutant bacteria deficient in 4EPS production exhibitedsignificantly less digging behavior (FIG. 8B).

Mice were habituated to a new cage with 20 marbles laid in a gridpattern. Number of marbles buried after 10 minutes was scored, and theresults are shown in FIG. 8C. The mice colonized with mutant bacteriadeficient in EPS production exhibited significantly less marble buryingbehavior (FIG. 8C).

These data show that co-colonizing mice with B. ovatus containing adeletion mutation in BO1194 (and therefore deficient in producing 4EPS)in accordance with some embodiments herein ameliorated symptomsassociated with anxiety and ASD in mice including repetitive behaviors,for example those measured by grooming, digging, and marble burying.

Example 9: Ultrasonic Vocalization Assay Behaviors of Mice Colonizedwith 4EP+/− Bacterial Pairs

An ultrasonic vocalization assay was performed as an assay ofcommunication behavior. Male mice colonized with engineered bacteriaeither lacking or producing 4EP were exposed to female mice for 15minutes daily for 5 days. On the 6^(th) day, each individual mouse wasintroduced to a novel female and frequency and duration of ultrasonicvocalizations was recorded for 3 minutes. The results are shown in FIGS.9A-B. Frequency (FIG. 9A) and duration (FIG. 9B) of the ultrasonicvocalization was significantly higher in the mice treated with B. ovatuscontaining a deletion mutation in BO1194 (and therefore deficient inproducing 4EPS), compared to controls treated with 4EPS-producingbacteria.

These data show that co-colonizing mice with B. ovatus containing adeletion mutation in BO1194 (and therefore deficient in producing 4EPS)in accordance with some embodiments herein ameliorated a symptomsassociated with anxiety and ASD in mice, including improvingcommunication behaviors, as measured by ultrasonic vocalization.

Example 10: Open Field Assay of Behaviors of Mice Colonized with 4EP+/−Bacterial Pairs

During open field assay, fecal pellets of mice colonized with engineeredbacteria either lacking or producing 4EP were counted. The 4EP-deficient(“4EP-”) males had significantly lower fecal output than the wild-typecontrol males (FIG. 10).

These data show that co-colonizing mice with B. ovatus containing adeletion mutation in BO1194 (and therefore deficient in producing 4EPS)in accordance with some embodiments herein lowered fecal output.

Example 11: Sulfation of 4EP and Modulation of the Same

Using R&D SYSTEMS Universal Sulfotransferase Assay Kit™, sulfation of4-ethylphenol (4EP) or para-cresol (PC) was determined colorimetrically(FIG. 11A). Samples from the assay were pooled and analyzed by liquidchromotography mass spectrometry (LCMS) for the confirmation that 4EPSand PCS are present (FIG. 11B). These experiments show that sulfated 4EPand PC can be detected in accordance with some embodiments herein.

An R&D SYSTEMS assay was used to determine whether quercetin andcurcumin can inhibit Sult1A1 (sulfotransferase family 1A member 1, anenzyme that catalyzes sulfate conjugation) modification of 4EP to 4EPS(FIG. 11C). These data show that quercetin and curcumin inhibitsulfation of 4EP in vitro. Accordingly, it is contemplated thatquercetin and curcumin in accordance with some embodiments herein caninhibit the production of 4EPS in a subject in need thereof.

SULT1a1 gene expression was measured in various tissues of micecolonized with engineered bacteria deficient in producing 4EPS (FIG.11D). It was observed that SULT1a1 is expressed in the GI tract of micecolonized with the bacteria.

4EP to 4EPS conversion was measured by cytosolic fractions from varioustissues of colonized mice (FIG. 11E). Substantial conversion of 4EP to4EPS was observed in the colon and liver.

In at least some of the previously described embodiments, one or moreelements used in an embodiment can interchangeably be used in anotherembodiment unless such a replacement is not technically feasible. Itwill be appreciated by those skilled in the art that various otheromissions, additions and modifications may be made to the methods,compositions, kits, and uses described herein without departing from thescope of the claimed subject matter. All such modifications and changesare intended to fall within the scope of the subject matter, as definedby the appended claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one of skill in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

Wherever a method of using a composition or product combination (e.g., acomposition or product combination comprising, consisting essentiallyof, or consisting of a bacteria and/or an antibiotic) is disclosedherein, the corresponding composition for use is also expresslycontemplated. For example, for the disclosure of a method of reducing orpreventing a symptom of anxiety and/or ASD in a selected subject,comprising administering an amount of a composition comprisinggenetically modified Bactericides ovatus, and/or genetically modifiedLactobacillus plantarum, and/or a polyphenol to the subject, thecorresponding composition comprising Bactericides ovatus and/orLactobacillus plantarum, and/or a polyphenol for use in reducing thelikelihood of, delaying the onset of, or ameliorating one or moresymptoms associated with anxiety and/or ASD is also contemplated.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those of skill in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

1. A composition or product combination comprising a geneticallyengineered Bacteroides ovatus comprising a loss-of-function mutation ina gene encoding BO1194, and/or a genetically engineered Lactobacillusplantarum comprising a loss-of function mutation in a gene encodingphenolic acid decarboxylase (PAD).
 2. The composition or productcombination of claim 1, wherein the composition or product combinationis formulated for oral or rectal administration to a mammalian subject.3. The composition or product combination of any one of claims 1-2,wherein the composition or product combination is selected from thegroup consisting of: a probiotic composition or product combination, apharmaceutical composition or product combination, a dietary supplement,and a food; or a combination of two or more of the listed items. 4.-29.(canceled)
 30. A method of reducing or inhibiting production of4-ethylphenol (4EP) and/or 4-ethylphenyl sulfate (4EPS) in a subject inneed thereof, the method comprising: administering to the subject acomposition or product combination comprising a genetically engineeredBacteroides ovatus comprising a loss-of-function mutation in a geneencoding BO1194, and/or a genetically engineered Lactobacillus plantarumcomprising a loss-of-function mutation in a gene encoding phenolic aciddecarboxylase (PAD); and permitting the genetically engineeredBacteroides ovatus and/or the genetically engineered Lactobacillusplantarum to proliferate in a gastrointestinal tract of the subject,whereby production of 4EP and/or 4EPS in the subject is reduced orinhibited.
 31. The method of claim 30, wherein reducing the levels of4EP and/or 4EPS ameliorates, delays the onset of, or decreases thelikelihood of a symptom associated with anxiety and/or ASD in thesubject.
 32. The method of any one of claims 30-31, further comprising,prior to said administering, selecting the subject as being within aclass of subjects that are in need of receiving the composition orproduct combination.
 33. The method of claim 32, wherein said selectingcomprises identifying the subject as having a symptom associated withanxiety and/or ASD. 34.-57. (canceled)